The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentration dependent loss of NADPH and reductase-supported or t-butylhydroperoxide-supported 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity of P450 2B1 with IC50 values of 39 and 210 (M, respectively. After incubation of P450 2B1 with 300 (M peroxynitrite, the heme moiety was not altered, but the protein was modified as shown by HPLC and spectral analysis. Western blot analysis of peroxynitrite-treated P450 2B1 demonstrated the presence of an extensive immunoreactive band after incubating with anti-nitrotyrosine antibody. However, the immunostaining was completely abolished after coincubation of the anti-nitrotyrosine antibody with 10 mM nitrotyrosine. These results indicated that one or more of the tyrosine residues in P450 2B1 were modified to nitrotyrosines. The decrease in enzymatic activity correlated with the increase in the extent of tyrosine nitration. Further d emonstratio n oftyrosine nitration was confirmed by GC-MS analysis by using 13C-labeled tyrosine and nitrotyrosine as internal standards, approximately 0.97 mol nitrotyrosine per mol P450 2B1 was found after treatment with peroxynitrite. The peroxynitrite-treated P450 2B1 was digested with Lys C and the resulting peptides separated by Tricine-SDS-PAGE. The amino acid sequence of the major nitrotyrosine-containing peptide corresponded to a peptide containing amino acid residues 160-225, which contains two tyrosine residues. Thus, incubation of P450 2B1 with peroxynitrite caused nitration of either tyrosine 190 or 203 of both of P450 2B1 concomitant with a loss of 2B1-dependent activity.